Combined chimeric antigen receptor targeting CD19 and CD20 and application thereof

ABSTRACT

The present invention provides a combined chimeric antigen receptor targeting CD19 and CD20 and application thereof. Specifically, the present invention provides a combined chimeric antigen receptor targeting CD19 and CD20, which comprises a scFv targeting CD19 and CD20, a hinge region, a transmembrane region, and an intracellular signaling domain. The present invention provides a nucleic acid molecule encoding the chimeric antigen receptor and a corresponding expression vector, a CAR-T cell, and applications thereof. The experimental results show that the chimeric antigen receptor provided by the present invention shows extremely high killing ability against tumor cells. The chimeric antigen receptor of the present invention targets CD19 and/or CD20 positive cells and can be used to treat CD19 and/or CD20 positive B-cell lymphoma, leukemia and other diseases.

TECHNICAL FIELD

The present invention relates to the field of bio-medicine, and moreparticularly to a combined chimeric antigen receptor targeting CD19 andCD20 and application thereof.

BACKGROUND

Malignant tumors of the blood system account for about 10% of humanmalignant tumors, and 95% of malignant tumors of the blood system arederived from B lymphocytes. Traditional chemotherapy and radiotherapyplay an important role in the treatment of malignant tumors of the bloodsystem. Some patients have significant effects, but it is difficult formost of the patients to be cured. New and effective treatments have beena hot topic in this field.

Adoptive T cell therapy has shown its powerful efficacy and brightprospect in the clinical treatment of malignant tumors. At present, itis regarded as one of the most promising methods for treatinghematological tumors. CD19 is highly expressed on the surface of mostB-cell malignancies. Multiple centers independently using ChimericAntigen Receptor (CAR)-modified T cells to target recurrent, refractorymalignant tumors of CD19-expressed B cell have achieved unprecedentedsuccess. At present, both of the two CAR-T products approved by FDA aretargeting CD19 antigen and their indications are also expanding, such aschronic lymphocytic leukemia. Although the efficacy of anti-CD19 CAR-Tis outstanding, many studies have shown that there are also manyproblems with CD19 chimeric antigen receptor (CAR) T cell therapy. Thereare still some patients with poor treatment results and easy to relapse.The reasons for this include the susceptibility of tumor cells toantigen escape. For example, a recent experiment of CD19 CAR-celltherapy showed that 90% of patients achieved complete remission, but 11%of these patients eventually relapsed, and the relapsing patients weremainly patients with CD19-negative tumor. In particular, in a clinicaltrial carried out at the University of Pennsylvania School of Medicineusing CART19 in the treatment of recurrent, refractory acute B-celllymphoma (R/R B-ALL), up to 94% of patients achieved complete remission.Although the initial response rate of this clinical trial was high,nearly 40% of patients relapsed after 1 month of treatment whichachieved complete remission, and more than 60% of relapsing patients hadCD19-negative tumor cells escape. Antigen escape has been found inadoptive transfer specific T cell receptors expressing NY-ESO1 andcancer vaccines treating melanoma. Spontaneous mutation and selectiveexpansion are the main reasons for antigen escape.

Therefore, there is an urgent need in the art to develop methods foreffectively treating tumors and preventing antigen escape.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method foreffectively treating tumors and preventing antigen escape.

An object of the present invention is to provide a combined chimericantigen receptor targeting CD19 and CD20 and preparation method thereof.

Specifically, it is an object of the present invention to provide asequence of the combined chimeric antigen receptor targeting CD19 andCD20 as well as a preparation method and activity identification of themodified T cell (CART-19/20) thereof. The present invention provides achimeric antigen receptor structure for use in the treatment of CD19 andCD20 positive B cell lymphoma.

In a first aspect of the invention, it provides a chimeric antigenreceptor (CAR), wherein the structure of the chimeric antigen receptoris shown in formula I as below:L-scFv1-I-scFv2-H-TM-C-CD3ζ  (I)

wherein,

each “-” is independently a linker peptide or a peptide bond;

L is an optional signal peptide sequence;

I is a flexible linker;

H is an optional hinge region;

TM is a transmembrane domain;

C is a co-stimulatory signaling molecule;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

one of scFv1 and scFv2 is an antigen binding domain targeting CD19, andthe other is an antigen binding domain targeting CD20.

In another preferred embodiment, the scFv1 is an antigen binding domaintargeting CD20, and the scFv2 is an antigen binding domain targetingCD19.

In another preferred embodiment, the structure of the antigen bindingdomain targeting CD20 is shown in formula A or B as below:V_(H1)-V_(L1) (A); V_(L1)-V_(H1) (B)

wherein V_(H1) is an anti-CD20 antibody heavy chain variable region;V_(L1) is an anti-CD20 antibody light chain variable region; and “-” isa linker peptide or a peptide bond.

In another preferred embodiment, the structure of the antigen bindingdomain targeting CD20 is shown in formula B.

In another preferred embodiment, the amino acid sequence of the V_(H1)is shown in SEQ ID NO 1, and the amino acid sequence of the V_(L1) isshown in SEQ ID NO 2; or

the amino acid sequence of the V_(H1) is shown in SEQ ID NO 3, and theamino acid sequence of the V_(L1) is shown in SEQ ID NO 4.

In another preferred embodiment, the V_(H1) and V_(L1) are linked with aflexible linker (or a linker peptide), and the flexible linker (or thelinker peptide) is 1-4, preferably 2-4, more preferably 3-4 consecutivesequences as shown in SEQ ID NO 7 (GGGGS).

In another preferred embodiment, the structure of the antigen bindingdomain targeting CD19 is shown in formula C or D as below:V_(L2)-V_(H2) (C); V_(H2)-V_(L2) (D)

wherein V_(L2) is an anti-CD19 antibody light chain variable region;V_(H2) is an anti-CD19 antibody heavy chain variable region; and “-” isa linker peptide or a peptide bond.

In another preferred embodiment, the structure of the antigen bindingdomain targeting CD19 is shown in formula D.

In another preferred embodiment, the amino acid sequence of the V_(L2)is shown in SEQ ID NO 5, and the amino acid sequence of the V_(H2) isshown in SEQ ID NO 6.

In another preferred embodiment, the V_(H2) and V_(L2) are linked with aflexible linker (or a linker peptide), and the flexible linker (or thelinker peptide) is 1-4, preferably 2-4, more preferably 3-4 consecutivesequence as shown in SEQ ID NO 7 (GGGGS).

In another preferred embodiment, the scFv1 and/or scFv2 aremouse-derived, humanized, humanized and mouse-derived chimeric, or fullyhumanized single chain antibody variable region fragments.

In another preferred embodiment, the structure of the chimeric antigenreceptor is shown in formula II as below:L-V_(L1)-V_(H1)-I-V_(H2)-V_(L2)-H-TM-C-CD3ζ  (II)

wherein each element is as described above.

In another preferred embodiment, the sequence of the flexible linker Icomprises 2-6, preferably 3-4 consecutive sequences as shown in SEQ IDNO 7 (GGGGS).

In another preferred embodiment, the L is a signal peptide of a proteinselected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, anda combination thereof.

In another preferred embodiment, the L is a signal peptide derived fromCD8.

In another preferred embodiment, the amino acid sequence of the L isshown in SEQ ID NO 8.

In another preferred embodiment, the H is a hinge region of a proteinselected from the group consisting of CD8, CD28, CD137, Ig4, and acombination thereof.

In another preferred embodiment, the H is a hinge region derived fromIg4.

In another preferred embodiment, the amino acid sequence of the H isshown in SEQ ID NO 9.

In another preferred embodiment, the TM is a transmembrane region of aprotein selected from the group consisting of CD28, CD3ε, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, and a combination thereof.

In another preferred embodiment, the TM is a transmembrane regionderived from CD8 or CD28.

In another preferred embodiment, the sequence of the TM is shown in SEQID NO 10 or 11.

In another preferred embodiment, the C is a co-stimulatory signalingmolecule of a protein selected from the group consisting of OX40, CD2,CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10,CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, and acombination thereof.

In another preferred embodiment, the C is a co-stimulatory signalingmolecule derived from 4-1BB or CD28.

In another preferred embodiment, the amino acid sequence of the C isshown in SEQ ID NO 12 or 13.

In another preferred embodiment, the amino acid sequence of the CD3ζ isshown in SEQ ID NO 14.

In another preferred embodiment, the amino acid sequence of the CAR isshown in SEQ ID NO 15 or 16.

In a second aspect of the invention, it provides a nucleic acidmolecule, encoding the chimeric antigen receptor of the first aspect ofthe invention.

In another preferred embodiment, the nucleic acid molecule is isolated.

In another preferred embodiment, the nucleotide sequence of the nucleicacid molecule is shown in SEQ ID NO 17 or 18.

In a third aspect of the invention, it provides a vector, comprising thenucleic acid molecule of the second aspect of the invention.

In another preferred embodiment, the vector comprises DNA and RNA.

In another preferred embodiment, the vector is selected from the groupconsisting of plasmid, virus vector, transposon, and a combinationthereof.

In another preferred embodiment, the vector comprises a DNA virus and aretrovirus vector.

In another preferred embodiment, the vector is selected from the groupconsisting of a lentiviral vector, an adenovirus vector, anadeno-associated virus vector, and a combination thereof.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect of the invention, it provides a host cell, comprisingthe vector of the third aspect of the invention or having the exogenousnucleic acid molecule of the second aspect of the invention integratedinto its genome or expressing the chimeric antigen receptor of the firstaspect of the invention.

In another preferred embodiment, the cell is an isolated cell.

In another preferred embodiment, the cell is a genetically engineeredcell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a CAR-T cell and/or aCAR-NK cell.

In another preferred embodiment, the cell targets both CD19 and CD20.

In a fifth aspect of the invention, it provides a method for preparing aCAR-T cell expressing the chimeric antigen receptor of the first aspectof the invention, wherein the method comprises the steps of: transducingthe nucleic acid molecule of the second aspect of the invention or thevector of the third aspect of the invention into a T cell, therebyobtaining the CAR-T cell.

In another preferred embodiment, the method further comprises the stepof detecting the function and effectiveness of the obtained CAR-T cell.

In a sixth aspect of the invention, it provides a preparation,comprising the chimeric antigen receptor of the first aspect of theinvention, the nucleic acid molecule of the second aspect of theinvention, the vector of the third aspect of the invention, or the hostcell of the fourth aspect of the invention, and a pharmaceuticallyacceptable carrier, diluent or excipient.

In another preferred embodiment, the preparation is a liquidpreparation.

In another preferred embodiment, the formulation of the preparation isan injection.

In another preferred embodiment, the preparation comprises the host cellof the fourth aspect of the invention, and the concentration of the hostcell is 1×10³-1×10⁸ cells/ml, preferably 1×10⁴-1×10⁷ cells/ml.

In a seventh aspect of the invention, it provides the use of thechimeric antigen receptor of the first aspect of the invention, thenucleic acid molecule of the second aspect of the invention, the vectorof the third aspect of the invention, or the host cell of the fourthaspect of the invention, for the preparation of a medicine or aformulation for preventing and/or treating tumor or cancer.

In another preferred embodiment, the tumor is selected from the groupconsisting of a hematological tumor, a solid tumor, and a combinationthereof; preferably, the tumor is a hematological tumor.

In another preferred embodiment, the blood tumor is selected from thegroup consisting of acute myeloid leukemia (AML), multiple myeloma (MM),chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL),diffuse large B cell lymphoma (DLBCL), and a combination thereof.

In another preferred embodiment, the solid tumor is selected from thegroup consisting of gastric cancer, peritoneal metastasis of gastriccancer, liver cancer, leukemia, renal cancer, lung cancer, smallintestine cancer, bone cancer, prostate cancer, colorectal cancer,breast cancer, large intestine cancer, cervical cancer, ovarian cancer,lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor,non-small cell lung cancer (NSCLC), glioma, endometrial cancer, and acombination thereof.

In an eighth aspect of the invention, it provides a kit for thepreparation of the cell of the fourth aspect of the invention, whereinthe kit comprises a container, and the nucleic acid molecule of thesecond aspect of the invention or the vector of the third aspect of theinvention located in the container.

In a ninth aspect of the invention, it provides a use of the cell of thefourth aspect of the invention, or the formulation of the sixth aspectof the invention for the prevention and/or treatment of cancer or tumor.

In a tenth aspect of the invention, it provides a method of treating adisease comprising administering an appropriate amount of the cell ofthe forth aspect of the invention, or the formulation of the sixthaspect of the invention, to a subject in need of treatment.

In another preferred embodiment, the disease is cancer or tumor.

It is to be understood that the various technical features of thepresent invention mentioned above and the various technical featuresspecifically described hereinafter (as in the Examples) may be combinedwith each other within the scope of the present invention to constitutea new or preferred technical solution, which needs not be described oneby one, due to space limitations.

DESCRIPTION OF DRAWINGS

FIG. 1 shows structure of the combined chimeric antigen receptortargeting CD19 and CD20. The structure of the CAR comprises a leadersequence, an antigen recognition sequence, a hinge region, atransmembrane region, a co-stimulatory factor signal region, and a CD3ζsignaling region.

FIG. 2 shows the detection of transfection efficiency of engineered Tcell with combined chimeric antigen receptors targeting CD19 and CD20.Protein L method was used to identify the expression level of CARgene-encoded protein on the surface of T cell membrane in CAR-T19/20scells cultured 7 days.

FIGS. 3A and 3B show the expression level of CD137 on the surface of Tcell membrane (FIG. 3A) and the secretion level of IFNγ in the culturesupernatant (FIG. 3B). Specifically, 1×10⁵ of CAR-T19/20s cells cultured7 days were taken and cultured respectively with CD19-positiveK562-CD19+ tumor cell line, CD20-positive K562-CD20+ tumor cell line,CD19 and CD20 double positive K562-CD19+CD20+ tumor cell line, RAJItumor cell line that naturally expresses CD19 and CD20, and CD19 andCD20 double negative K562 tumor cell line, or without tumor cells, in200 μl of GT-551 medium for 18 h with a ratio of 1:1. Then theexpression level of CD137 on the surface of T cell membrane and thesecretion level of IFNγ in the culture supernatant were detectedrespectively.

FIG. 4 shows the detection of tumor-killing activity of CAR-T19/20scells, mainly by detecting the secretion level of LDH in the supernatantafter co-culture. Specifically, 1×10⁴ cells of CD19-positive K562-CD19+tumor cell line, CD20-positive K562-CD20+ tumor cell line, CD19 and CD20double positive K562-CD19+CD20+ tumor cell line, RAJI or RAMOS tumorcell line that naturally expresses CD19 and CD20, or CD19 and CD20double negative K562 tumor cell line were co-cultured with thecorresponding T cells, respectively, in 100 μl of GT-551 medium for 8 hwith a ratio as shown in the figure. Then the secretion level of LDH wasdetected, and this figure shows the statistical analysis results of thepercentages of LDH release in corresponding co-culture samples.

FIG. 5 shows the detection of tumor-killing activity of CAR-T19/20scells, mainly by detecting the expression level of CD107a on the surfaceof T cell membrane. Specifically, 1×10⁵ CAR-T19/20s cells were taken andcultured respectively with CD19-positive K562-CD19+ tumor cell line,CD20-positive K562-CD20+ tumor cell line, CD19 and CD20 double positiveK562-CD19+CD20+ tumor cell line, RAJI or RAMOS tumor cell line thatnaturally expresses CD19 and CD20, and CD19 and CD20 double negativeK562 tumor cell line, or without tumor cells, in 200 μl of GT-551 mediumfor 4 h with a ratio of 1:2. Then the expression level of CD137 on thesurface of T cell membrane was detected respectively.

FIGS. 6A-6F shows the results of CAR-T19/20s cells killing tumor cellsin RAJI-Luc/NSG leukemia model mice. FIG. 6A shows the detection of thetransfection efficiency of CAR-T19/20s cells. Protein L method was usedto identify the expression level of CAR gene-encoded protein on thesurface of T cell membrane in CAR-T19/20s cells cultured 9 days. 1×10⁵CAR-T19/20s cells cultured 10 days (FIG. 6B) and 18 days (FIG. 6C) weretaken and cultured respectively with CD19-positive K562-CD19+ tumor cellline, CD20-positive K562-CD20+ tumor cell line, CD19 and CD20 doublepositive K562-CD19+CD20+ tumor cell line, RAJI or RAMOS tumor cell linethat naturally expresses CD19 and CD20, and CD19 and CD20 doublenegative K562 or MOLT4 tumor cell line, or without tumor cells, in 200 lof GT-551 medium for 18 h with a ratio of 1:1. Then the expression levelof CD137 on the surface of T cell membrane was detected respectively.FIG. 6D shows the secretion level of IFNγ in the culture supernatant.FIG. 6E shows the average body weight changes and average fluorescenceintensity changes of mice injected with CAR-T19/20s cells within 21days, which are recorded every 7 days. FIG. 6F shows in vivo imaging ofmice injected with CAR-T19/20s cells on day 0 (DO), 7 (D7), 14 (D14),and 21 (D21) after injection.

FIGS. 7A-7C show the preliminary functional verification results ofCAR-T20.1, CAR-T20.2, and CAR-T20.4.

FIG. 7A shows the testing results of the transfection efficiency of Tcells. The expression level of CAR gene-encoded protein on the surfaceof T cell membrane in CAR-T20s cells cultured 7 days was identified bythe DNA copy number detection method. FIG. 7B shows the secretion levelof IFNγ in the supernatant of CAR-T20s cells co-cultured with targetcells. FIG. 7C shows the expression level of CD137 on the surface of Tcell membranes after co-culture.

FIGS. 8A-8C show the functional verification results of CAR-T20.5,CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10.

FIG. 8A shows the testing results of the transfection efficiency of Tcells. The expression level of the CAR gene-encoded protein on thesurface of the T cell membrane in CAR-T20s cells cultured 7 days wasidentified by the Protein L method. FIG. 8B shows the secretion level ofIFNγ in the supernatant of CAR-T20s cells co-cultured with target cells.FIG. 8C shows the expression level of CD137 on the surface of T cellmembranes after co-culture.

FIGS. 9A-9C show the functional verification results of CAR-T20.11,CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15 and CAR-T20.16. FIG. 9Ashows the testing results of the transfection efficiency of T cells. Theexpression level of the CAR gene-encoded protein on the surface of the Tcell membrane in CAR-T20s cells cultured 7 days was identified by theProtein L method. FIG. 9B shows the secretion level of IFNγ in thesupernatant of CAR-T20s cells co-cultured with target cells. FIG. 9Cshows the expression level of CD137 on the surface of T cell membranesafter co-culture.

FIGS. 10A-10D show the functional verification results of CAR-T20.17,CAR-T20.18 and CAR-T20.19.

FIG. 10A shows the detection results of T cell transfection efficiency.FIG. 10B shows the IFNγ secretion level in the supernatant afterco-culture. FIG. 10C shows the detection of tumor cell killing abilityof CAR-T20s cells, mainly by detecting the secretion level of LDH in thesupernatant after co-culture. FIG. 10D shows the expression level ofCD137 on the surface of T cell membranes.

FIG. 11 shows the inhibitory effect of CAR-T19/20s cells on transplantedtumor cells in mice.

FIG. 12 shows the experimental process of the phase I clinical trial ofCART-TN-20OF-19.

FIG. 13 shows the detection results of the CAR copy number.

FIG. 14 shows the change in tumor size of a subject.

In the figures, TN-OF-19 and TN-20OF-19 have the same meaning, andTN-LEU-19 and TN-20 LEU-19 have the same meaning, both indicating CARTcells having the corresponding CAR structure.

MODES FOR CARRYING OUT THE PRESENT INVENTION

After extensive and intensive studies, the inventors unexpectedlyobtained a CAR-T cell that simultaneously targets CD19 and CD20.Specifically, the present invention provides a chimeric antigen receptorthat simultaneously targets CD19 and CD20, which comprises a signalpeptide, an anti-CD20 scFv, an anti-CD19 scFv, a hinge region, atransmembrane region, and an intracellular T cell signaling region.Moreover, the anti-CD20 scFv and anti-CD19 scFv were obtained through alarge number of screenings, which were linked with peptide fragment withmultiple repeat structure (G4S). The CAR-T cells of the presentinvention can recognize both CD19 and CD20 antigens at the same time,reducing the risk of immune escape caused by down-regulation or deletionof antigen expression during the treatment of single-target CAR-T cells.Compared to CAR-T cells targeting single antigens and other doubletarget CAR-T cells (targeting CD19 and CD20), the CAR-T cells of thepresent invention that simultaneously recognize two targets havestronger killing ability against tumor cells, less cytotoxicity, lowerside effects, wider treatment range, lower recurrence rate and betterefficacy. The present invention has been completed on the basis of this.

Terms

To make the disclosure easier to understand, some terms are firstlydefined. As used in this application, unless expressly stated otherwiseherein, each of the following terms shall have the meanings given below.Other definitions are set forth throughout the application.

The term “about” may refer to a value or composition within anacceptable error range for a particular value or composition asdetermined by those skilled in the art, which will depend in part on howthe value or composition is measured or determined.

The term “administering” refers to the physical introduction of aproduct of the invention into a subject using any one of various methodsand delivery systems known to those skilled in the art, includingintravenous, intramuscular, subcutaneous, intraperitoneal, spinal orother parenteral administration, such as by injection or infusion.

The term “antibody” (Ab) may comprise, but is not limited to, animmunoglobulin that specifically binds an antigen and contains at leasttwo heavy (H) chains and two light (L) chains linked by disulfide bonds,or an antigen binding parts thereof. Each H chain contains a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region contains three constant domains,CH1, CH2, and CH3. Each light chain contains a light chain variableregion (abbreviated herein as VL) and a light chain constant region. Thelight chain constant region contains a constant domain CL. The VH and VLregions can be further subdivided into hypervariable regions calledcomplementarity determining regions (CDR), which are interspersed withinmore conservative regions called framework regions (FR). Each VH and VLcontains three CDRs and four FRs, which are arranged from amino terminalto carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen.

CD20

Although the efficacy of anti-CD19 CAR-T is outstanding, many studieshave shown that there are still many problems with CD19 chimeric antigenreceptor (CAR) T cell therapy. There are still some patients with poortreatment results and easy to relapse. This includes the susceptibilityof tumor cells to antigen escape.

In order to prevent the escape of CD19 CAR-T antigen, the inventorsdesigns a combined bispecific CAR (ie, BICAR) that targets both CD19 andCD20, so that when CD19 antigen escapes and is not expressed in tumorcells, CD20 can be recognized to clear tumor cells in vivo.

CD20 is expressed in most patients with B-cell acute lymphoblasticleukemia, including some CD19 negative patients after anti-CD19 CAR-Ttreatment. CD20 is a glycosylated protein, and is the first identified Bcell membrane marker. CD20 is also known as B 1, and encoded by the MS4Agene. CD20 molecule has four transmembrane hydrophobic regions, and itsN-terminal and C-terminal are located on the cytoplasmic side, formingtwo closed loops outside the cell, and respectively called big loop andsmall loop. CD20 is specifically expressed in more than 95% of normaland cancerous B cells. These cells are in the pre-B cell stage andsubsequent developmental stages, and CD20 stops expression until thecells differentiated into plasma cells. The present invention uses CD20as another target for immunotherapy of B cell malignancies.

Bispecific Chimeric Antigen Receptor Targeting CD19 and CD20

Cellular immunotherapy is an emerging and highly effective tumortreatment model, and is a new type of autoimmunolgy treatment forcancer. It is a method for in vitro culture and amplification of immunecells collected from a patient using biotechnology and biologicalagents, and then the cells are transfused back to the patient tostimulate and enhance the body's autoimmune function, thereby achievingthe purpose of treating tumors. The skilled in the art have been workingto develop new cellular immunotherapy to increase the efficiency andreduce the side effect.

The present invention proposes a rational and optimized single-chaindesign and system, that is, combined bispecific CAR, which can beeffectively integrated into primary human T cells, and cansimultaneously target CD19 and CD20 when the T cells are activated. TheCAR-T cells of the invention are capable of recognizing two antigens(CD19 and CD20). The invention provides a very effective potentialmethod for preventing antigen escape.

The present invention uses CAR that simultaneously targets CD19 andCD20. Compared with CARs that target a single antigen, the affinity isenhanced, the activity of T cells is increased, and the targets have anadditive or synergistic effect. In addition, due to uneven expressionlevels of CD19 and CD20 in tumor cells, double target CAR-T therapy hasa wider scope. The CAR-T that simultaneously targets CD19 and CD20 onthe surface of tumor cells can reduce the possibility of antigen escapecaused by down-regulation or deletion of single surface antigen.

Bispecificity means that the same CAR can specifically bind andimmunorecognize two different antigens, and the CAR can generate animmune response by binding to any one of the antigens.

The CD19 and CD20 bispecific CAR of the present invention has a singlestructure and comprises anti-CD19 and anti-CD20 scFvs. Wherein, the CARcomprises a CD19 scFv and a CD20 scFv, and the amino acid sequences,sequencing and hinge of CD19 scFv and CD20 scFv are the main factorsaffecting its function.

Specifically, the chimeric antigen receptor (CAR) of the inventioncomprises an extracellular domain, a transmembrane domain, and anintracellular domain. The extracellular domain comprises atarget-specific binding element (also known as an antigen bindingdomain). The intracellular domain comprises a co-stimulatory signalingregion and a ζ chain. The co-stimulatory signaling region refers to apart of the intracellular domain that comprises a co-stimulatorymolecule. The co-stimulatory molecule is a cell surface moleculerequired for efficient response of lymphocytes to antigens, rather thanan antigen receptor or ligand thereof.

A linker can be incorporated between the extracellular domain and thetransmembrane domain of the CAR, or between the cytoplasmic domain andthe transmembrane domain of the CAR. As used herein, the term “linker”generally refers to any oligopeptide or polypeptide that plays a role oflinking the transmembrane domain to the extracellular domain or thecytoplasmic domain in a polypeptide chain. The linker may comprise 0-300amino acids, preferably 2-100 amino acids and most preferably 3-50 aminoacids.

In a preferred embodiment of the invention, the extracellular domain ofthe CAR provided in the present invention comprises an antigen bindingdomain targeting CD19 and CD20. When the CAR of the present invention isexpressed in T cell, antigen recognition can be performed based onantigen binding specificity. When the CAR binds to its associatedantigen, it affects tumor cell, causing tumor cell to fail to grow, todeath or to be affected otherwise, causing the patient's tumor burden toshrink or eliminate. The antigen binding domain is preferably fused tothe intracellular domain from one or more of the co-stimulatory moleculeand the ζ chain. Preferably, the antigen binding domain is fused with anintracellular domain of a combination of a 4-1BB signaling domain and aCD3ζ signaling domain.

As used herein, the “antigen binding domain” and “single-chain antibodyfragment” refer to an Fab fragment, an Fab′ fragment, an F (ab′) 2fragment, or a single Fv fragment that has antigen-binding activity. TheFv antibody contains the heavy chain variable region and the light chainvariable region of the antibody, but has no constant region. The Fvantibody has the smallest antibody fragment with all antigen-bindingsites. Generally, Fv antibodies also comprise a polypeptide linkerbetween the VH and VL domains, and can form the structure required forantigen binding. The antigen binding domain is usually a scFv(single-chain variable fragment). The size of scFv is typically ⅙ of acomplete antibody. The single-chain antibody is preferably an amino acidchain sequence encoded by a nucleotide chain. As a preferred embodimentof the present invention, the scFv comprises antibodies thatspecifically recognize CD19 and CD20.

As for the hinge region and the transmembrane region (transmembranedomain), the CAR can be designed to comprise a transmembrane domainfused to the extracellular domain of the CAR. In one embodiment, atransmembrane domain that is naturally associated with one of thedomains in the CAR is used. In some embodiments, transmembrane domainsmay be selected or modified by amino acid substitutions to avoid bindingsuch domains to the transmembrane domain of the same or differentsurface membrane proteins, thereby minimizing the interaction with othermembers of the receptor complexes.

The intracellular domain in the CAR of the invention comprises thesignaling domain of 4-1BB and the signaling domain of CD3ζ.

Preferably, the CAR structure of the present invention, in turn,comprises a signal peptide sequence (also known as leader sequence), anantigen recognition sequence (antigen-binding domain), a hinge region, atransmembrane region, a co-stimulatory factor signal region, and aCD3zeta signaling region (ζ chain portion). The order of connection isshown in FIG. 1.

In another preferred embodiment, the present CAR is TN-LEU-19. Theantigen binding domain targeting CD20 comprises a heavy chain sequence(SEQ ID NO 1) and a light chain (VL) sequence (SEQ ID NO 2) of thesingle-chain variable region derived from Leu16 antibody.

Heavy chain sequence of single-chain variableregion (VH) derived from Leu16 antibody: (SEQ ID NO 1)EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSS Light chain sequence of single-chain variableregion (VL) derived from Leu16 antibody: (SEQ ID NO 2)DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGG TKLEIK

In another preferred embodiment, the present CAR is TN-OF-19. Theantigen binding domain targeting CD20 comprises a heavy chain sequence(SEQ ID NO 3) and a light chain sequence (SEQ ID NO 4) of thesingle-chain variable region derived from Ofatumumab antibody.

Heavy chain sequence of single-chain variableregion (VH) derived from Ofatumumab antibody: (SEQ ID NO 3)EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS Light chain sequence of single-chain variableregion (VL) derived from Ofatumumab antibody: (SEQ ID NO 4)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQ GTRLEIK

In another preferred embodiment, the antigen-binding domain targetingCD19 in the CAR of the present invention comprises a light chain (VL)sequence (SEQ ID NO 5) and a heavy chain sequence (SEQ ID NO 6) of thesingle-chain variable region derived from FMC63 antibody.

Amino acid sequence of the light chain of single-chain variable region(VL) derived from FMC63 antibody: (SEQ ID NO 5)DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITNucleotide sequence of the light chain of single-chain variable region(VL) derived from FMC63 antibody: (SEQ ID NO 21)gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300gggaccaagc tggagatcac a 321Amino acid sequence of the heavy chain of single-chain variable region(VH) derived from FMC63 antibody: (SEQ ID NO 6)EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSNucleotide sequence of the heavy chain of single-chain variable region(VH) derived from FMC63 antibody: (SEQ ID NO 22)gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360

Specifically, the sequences of other elements in the CAR of the presentinvention are as follows:

The leader sequence is the leader sequence of CD8 antigen: (SEQ ID NO 8)MALPVTALLLPLALLLHAARP

The linker sequences (i.e., flexible linker I) between the heavy chainand light chain of the single-chain variable region are:

Amino acid sequence: (SEQ ID NO 19) GGGGSGGGGSGGGGSNucleic acid sequence: (SEQ ID NO 20)ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctThe hinge region is selected from the sequence of IgG4 Hinge-CH2-CH3:(SEQ ID NO 9) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

The transmembrane region is the transmembrane region sequence of CD8(CD8TM) or CD28 (CD28TM) antigen:

CD8TM: (SEQ ID NO 10) IYIWAPLAGTCGVLLLSLVITLYC CD28TM: (SEQ ID NO 11)FWVLVVVGGVLACYSLLVTVAFIIFWV

The co-stimulatory factor signal region is derived from the sequence of4-1BB or CD28 cytoplasmic signaling motif:

4-1BB: (SEQ ID NO 12) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD28:(SEQ ID NO 13) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

The signaling region of CD3ζ is derived from the sequence ofimmunorecceptor tyrosine-based activation motif (ITAM) of CD3ζ in theTCR complex:

(SEQ ID NO 14) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR

In a preferred embodiment, the complete nucleic acid sequences and aminoacid sequences of the two CARs constructed in the present invention areas follows:

The complete nucleic acid sequence of TN-OF-19 is as follows:(SEQ ID NO: 18) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGThe complete amino acid sequence of TN-OF-19 is as follows:(SEQ ID NO: 16) MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRThe complete nucleic acid sequence of TN-LEU-19 is as follows:(SEQ ID NO: 17) ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAATCTCCAGCTATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAATTACATGGACTGGTACCAGAAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTTTTAATCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGACTATTACTGTGCAAGATCTAATTATTACGGTAGTAGCTACTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGGCAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGACATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGThe complete amino acid sequence of TN-LEU-19 is as follows:(SEQ ID NO: 15) METDTLLLWVLLLWVPGSTGDIVLTQSPAILSASPGEKVIMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR

The Design of the BICAR of the Present Invention has the FollowingAdvantages:

First, CD19 and CD20 are expressed in most malignant B-cell tumors.Secondly, in general, when expanding CAR structure to increase T cellrecognition ability, problems such as increased adverse targeting,increased cytotoxicity, and increased side effects are oftenencountered. However, this is not the case for CD19 and CD20 becauseboth of them are only expressed in B cells with the same tumor toxicitycurve. Finally, the expression of CD19 and CD20 in B cells can promotethe survival of B cells. And the loss of both antigens during treatmentis a very low probability event. Therefore, targeting CD19 and CD20 isexpected to provide effective prevention of antigen escape of malignantB cells.

Compared with the single CAR of CD19 or CD20, BICAR has the followingadvantages:

First, compared with expressing two independent CARs, when expressingBICAR in a single T cell, the DNA footprint is significantly reduced(the DNA length is reduced by 40%). The size and the length of structurecan significantly affect the packaging and transduction efficiency ofthe viral vector, thus directly affecting the clinical efficacy.Secondly, compared to the mixture of two different single CARs, BICARcan significantly reduce the cost of treatment (BICAR is completelycompatible with the current T cell production process without addingadditional burden) and increase the clinical cure rate. Finally, CD19and CD20 have been verified in a large number of clinical studies andare relatively safe.

In the present invention, we constructed two types of chimeric antigenreceptor structures (TN-LEU-19, TH-OF-19) targeting CD19 and CD20 basedon the sequence of CD19 mouse-derived monoclonal antibody FMC63 and thesequences of CD20 mouse-derived monoclonal antibody leu-16 andOfatumumab. We completed the analysis and identification of theexpression levels, in vitro activation capacity, and tumor cell killingefficacy of these two chimeric antigen receptors in primary T cells.Finally, it was found that the T-cells modified with TN-LEU-19 orTH-OF-19 chimeric antigen receptors have a strong ability to kill invitro and to clear malignant tumors carrying CD19 and CD20 positiveantigens in vivo, and Ofatumumab is better than leu16. This provides anew effective method and preparation for the clinical application ofCAR-T in the treatment of CD19 and CD20-positive leukemias andlymphomas.

The present invention designed and optimized single-specific anddouble-specific CARs. These CARs have a powerful killing ability againstB-cell malignancies expressing CD19 or CD20. BICAR allows a singleT-cell product to target two clinically validated antigens associatedwith B-cell leukemia and lymphoma, ultimately reducing the risk of tumorrecurrence due to the loss or escape of a single antigen. The inventioncan be further used in the design of new BICAR, thus increasing theantigen's applicability and increasing the efficacy of T cell therapyfor cancer.

Chimeric Antigen Receptor T Cell (CAR-T Cell)

As used herein, the terms “CAR-T cell”, “CAR-T”, “CART”, “CAR-T cell ofthe present invention” all refer to the CAR-T cell that targets bothCD19 and CD20 of the forth aspect of the invention. Specifically, theCAR structure of the CAR-T cells comprises an anti-CD19 scFv, ananti-CD20 scFv, a hinge region, a transmembrane region, and anintracellular T cell signaling region in turn, wherein the anti-CD20scFv and anti-CD19 scFv are linked with a peptide having multiplerepeating structures (G4S). Compared with CAR-T targeting a singleantigen, the CAR-T cell that simultaneously recognizes two targets aremore lethal and have a wider range of treatment.

Vector

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to comprise the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically.

The present invention also provides vectors in which the expressioncassette of the present invention is inserted. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral vectorshave the advantage over vectors derived from onco-retroviruses such asmurine leukemia viruses in that they can transduce non-proliferatingcells, such as hepatocytes. They also have the advantage of lowimmunogenicity.

In brief summary, the expression cassette or nucleic acid sequence ofthe invention is typically and operably linked to a promoter, andincorporated into an expression vector. The vectors can be suitable forreplication and integration in eukaryotes. Typical cloning vectorscontain transcription and translation terminators, initiation sequences,and promoters useful for regulation of the expression of the desirednucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immune and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest comprise expressionvectors, replication vectors, probe generation vectors, and sequencingvectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al, (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors comprise, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters,inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters comprise, but are not limited to a metallothionein promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a ceil can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers comprise, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may comprise genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellcomprise calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell comprise the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cellcomprise colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids comprise the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviralvector.

Preparation

The invention provides a preparation comprising the CAR-T cell of theforth aspect of the invention, and a pharmaceutically acceptablecarrier, diluent or excipient. In one embodiment, the preparation is aliquid preparation. Preferably, the preparation is an injection.Preferably, the concentration of the CAR-T cells in the preparation is1×10³-1×10⁸ cells/ml, more preferably 1×10⁴-1×10⁷ cells/ml.

In one embodiment, the preparation may comprises buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. The preparation of the invention ispreferably formulated for intravenous administration.

Therapeutic Application

The invention comprises therapeutic applications using cells (e.g., Tcells) transduced with a lentiviral vector (LV) encoding the expressioncassette of the invention. The transduced T cells can target the tumorcell markers CD19 and CD20, synergistically activate T cells, and causeT cell immune responses, thereby significantly increasing the killingefficiency against tumor cells.

Thus, the present invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a CAR-T cellof the invention.

In one embodiment, the present invention comprises a class of celltherapies, wherein T cells from autologous patient (or heterologousdonor) are isolated, activated and genetically modified to generateCAR-T cells, and then injected into the same patient. The probability ofgraft versus host disease in this way is extremely low, and antigens arerecognized by T cells in a non-MHC-restricted manner. In addition, oneCAR-T can treat all cancers that express the antigen. Unlike antibodytherapies, CAR-T cells are able to replicate in vivo resulting inlong-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In addition, the CAR mediated immune response may be part of an adoptiveimmunotherapy approach in which CAR-modified T cells induce an immuneresponse specific to the antigen binding moiety in the CAR. For example,an anti-CD19CD20 CAR-T cell elicits an immune response specificallyagainst cells expressing CD19 and CD20.

Although the data disclosed herein specifically discloses lentiviralvector comprising CD19CD20 scFv, hinge and transmembrane domain, and4-1BB and CD3ζ signaling domains, the invention should be construed tocomprise any number of variations for each of the components of theconstruct as described elsewhere herein.

Cancers that may be treated comprise tumors that are unvascularized orlargely unvascularized, and tumors that are vascularized. Cancers maycomprise non-solid tumors (such as hematological tumors, for example,leukemias and lymphomas) or solid tumors. Types of cancers to be treatedwith the CARs of the invention comprise, but are not limited to,carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies e.g.,sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also comprised.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers comprise leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, comprise fibrosarcoma,myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreaticcancer and ovarian cancer.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expaning the cells, ii) introducing a nucleic acid encoding a CAR to thecells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

The present invention provides methods for treating tumors comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2, IL-17 or othercytokines or cell populations. Briefly, pharmaceutical compositions ofthe present invention may comprise a target cell population as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J.of Med. 319: 1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermaliy, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunotherapeutic agents. In a further embodiment,the cell compositions of the present invention are administered to apatient in conjunction with (e.g., before, simultaneously or following)bone marrow transplantation, or the use of chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide.For example, in one embodiment, subjects may undergo standard treatmentwith high dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for patientadministration can be performed according to art-accepted practices. Ingeneral, 1×10⁶ to 1×10¹⁰ of the modified T cells of the invention (e.g.,CAR-T-19/20 cells) can be applied to patients by means of, for example,intravenous infusion each treatment or each course of treatment.

The Advantages of the Present Invention are

(1) As for the chimeric antigen receptor of the present invention, theextracellular antigen binding domain is specific anti-CD20 scFv andanti-CD19 scFv; the CAR formed by combining the specific anti-CD20 scFvand anti-CD19 scFv to a specific hinge region and an intracellulardomain shows a great ability of killing tumor cells with lowcytotoxicity and low side effects.

(2) The chimeric antigen receptor provided by the invention can achievestable expression and membrane localization of CAR protein after T cellsare infected by lentivirus carrying CAR gene.

(3) The CAR-modified T cell of the present invention has a longersurvival time in vivo and strong anti-tumor efficacy; the optimized CARwith the IgG4 Hinge-CH2-CH3 linker region can avoid the binding of theFc receptor and the subsequent ADCC effect (antibody-dependentcytotoxicity).

(4) Compared with two independent CARs, the bispecific chimeric antigenreceptor of the present invention comprises both anti-CD20 scFv andanti-CD19 scFv, and the DNA footprint is significantly reduced (the DNAlength is reduced by 40%), and the size of structure is small, which isbeneficial for the packaging and transduction efficiency of viralvectors, thus directly improving clinical efficacy. In addition, thebispecific CAR of the invention has lower cost, higher cure rate, andmore safety.

(5) The T-cells modified with TN-LEU-19 or TH-OF-19 chimeric antigenreceptors of the present invention have very strong ability to kill invitro and to clear malignant tumors carrying CD19 and CD20 positiveantigens in vivo, and Ofatumumab is stronger. This provides a neweffective method and preparation for the clinical application of CAR-Tin the treatment of CD19 and CD20-positive leukemias and lymphomas.

(6) The CAR-T cells of the present invention have a killing effect onmost malignant B-cell tumors, have a wider treatment range and a largercoverage rate, and can more effectively prevent tumor cells fromescaping.

The present invention will be further illustrated below with referenceto the specific examples. It is to be understood that these examples arefor illustrative purposes only and are not intended to limit the scopeof the invention. The experimental methods with no specific conditionsdescribed in the following examples are generally performed under theconventional conditions, or according to the manufacturer'sinstructions. Percentages and parts are by weight unless otherwisestated.

Example 1 Construction of Lentiviral Expression Vector

The full-length DNA was synthesized and cloned to achieve theconstruction of encoding plasmids. The pWPT lentiviral vector wasselected as a cloning vector, and the cloning sites were BamH I and SalI sites. Wherein, the structures of the two CARs designed in the presentinvention are shown in FIG. 1. The nucleic acid sequence of TN-LEU-19 isshown in SEQ ID NO 17, and the nucleic acid sequence of TN-OF-19 isshown in SEQ ID NO 18.

Example 2 Preparation of CAR-T Cells

(1) Mononuclear cells (PBMCs) were isolated from venous blood of healthypeople by density gradient centrifugation.

(2) On day 0, PBMCs were seeded in a cell culture flask previouslycoated with CD3 monoclonal antibody (OKT3) at a final concentration of 5μg/mL and Retronectin (purchased from TAKARA) at a final concentrationof 10 μg/mL. The medium was GT-551 cell culture medium containing 1%human albumin. Recombinant human interleukin 2 (IL-2) was added to themedium at a final concentration of 1000 U/mL. The cells were cultured inan incubator with a saturated humidity and 5% CO₂ at 37° C.

(3) On day 1, the supernatant of the PBMCs culture was slowly aspiratedand discarded. New GT-551 cell culture medium containing 1% humanalbumin was added, and recombinant human interleukin 2 (IL-2) was addedto the medium at a final concentration of 1000 U/mL. The cells werecontinuously cultured in an incubator with a saturated humidity and 5%CO₂ at 37° C.

(4) On day 3, fresh medium, concentrated and purified TN-LEU-19 orTN-OF-19 lentivirus solution, protamine sulfate (12 ug/ml), and IL-2 (ata final concentration of 1000 U/mL) were added. After 12 hours ofinfection in a 5% CO₂ incubator at 37° C., the culture medium wasdiscarded, and fresh medium was added, and cultivation was continued ina 5% CO₂ incubator at 37° C.

(5) Starting from day 6, CAR-T19/20s (CART-TN-OF-19 and CART-TN-LEU-19)cells can be taken for corresponding activity assay.

Example 3 Detection of the Integration Rate of the CAR Gene in the TCell Genome and the Expression Level of the Encoded Protein Thereof onthe Membrane Surface

0.5×10⁶ CAR-T19/20s cell sample cultured 7 days in Example 2 were takenrespectively to test the transfection efficiency of the combinedchimeric antigen receptor engineered T cells targeting CD19 and CD20.Protein L method was used to identify the expression level of CARgene-encoded protein on the surface of T cell membrane in CAR-T19/20scells cultured 7 days.

The result is shown in FIG. 2, and two CAR structures (FIG. 1) designedin the present invention can be expressed in their correspondingmodified T cells and complete the cell membrane surface localization.

Example 4 Detection of the In Vitro Activation Ability of CAR-T19/20s

Cell activation level indicator proteins CD137 and IFNγ was detectedusing CAR-T19/20s cells cultured 7 days in Example 2. 1×10⁵ ofCART-T19/20 cells cultured 7 days were taken and cultured respectivelywith CD19, CD20-positive K562−CD19+, K562−CD20+, K562−CD19+CD20+ andRaji (naturally expressing CD19 and CD20) tumor cell line, as well asCD19CD20-negative K562 tumor cell line, or without tumor cells, in 200μl of GT-551 medium for 18 h with a ratio of 1:1. Then the expressionlevel of CD137 on the surface of T cell membrane and the secretion levelof IFNγ in the culture supernatant were detected respectively.

The results are shown in FIGS. 3A and 3B, the expressions of CD137 onthe surface of two CART cells were detected, and the expressions of IFNγin the culture supernatants were detected. Wherein, TN-OF-19 has higherCD137 activation level and IFNγ release level than TN-LEU-19.

Example 5 Detection of LDH Levels Released by CAR-T19/20s Cells forKilling Targeted Tumors In Vitro

The CAR-T19/20s cells prepared in Example 2 were tested as follows:

Experimental wells, effector cell control wells, target cell controlwells, target cell maximum release wells, medium control wells, andvolume control wells (target cells comprise CD19, CD20 positiveK562-CD19, K562-CD20, K562-CD19-CD20, and Raji cells; and effector cellscomprise NT, CART-TN-LEU-19, and CART-TN-OF-19) were set.

The effect-target ratio was set, wherein number of effector cells:numberof target cells=5:1, 10:1, 20:1, and 40:1. Number of cells: 1×10⁴ oftarget cells, 50 ul/well; and effective cells are 5×10⁴ cells/well,1×10⁵ cells/well, 2×10⁵ cells/well, and 5×10⁵ cells/well. Differentdilution ratios of effector cells and target cells were added to theexperimental wells with an effect-target ratio of 5:1, 10:1, 20:1, and40:1. A total of 100 ul of the two cells (50 ul of effector cells+50 ulof target cells) were added to the cell culture plate, wherein 3 repeatswere set. As for effector cell control wells, i.e. effector cells:targetcells=5:0, 10:0, 20:0, and 40:0, 5×10⁴/well, 10⁵/well, 2×10⁵/well,5×10⁵/well of effector cells and 50 ul medium were added, wherein 2repeats were set. As for target cell control wells, 1×10⁴/well, 50 ul oftarget cell and 50 ul medium were added. As for target cells maximumrelease wells, 1×10⁴, 50 ul of target cells and 50 ul medium were added.10 ul lysate was added after incubating for 3 h and 15 min. As for themedium control wells, 100 ul medium was added. As for the volume controlwell, 100 ul medium was added.

After incubating for 3 h and 15 min, while adding 10 ul of lysate to thetarget cell maximum release wells, 10 ul of lysate was added andincubated at 37° C. The mixture was centrifuged at 250 g for 4 min and50 ul/well of cell supernatant was transferred to a new enzyme plate. 50ul/well of substrate mixture was added (kept in dark, with 12 ml ofdetection buffer added to a bottle of substrate mixture). The mixturewas incubated at room temperature in dark for 30 min. Finally, 50ul/well stop solution was added. The plate was read at 490 nm in 1 h,and the data was analyzed.

The results are shown in FIG. 4. Both CART cells can well induceapoptosis and release LDH in BCMA-positive tumor cells. Wherein,CART-TN-LEU-19 can better kill CD19CD20 positive tumor cells, andrelease higher LDH and the like than CART-TN-OF-19.

Example 6 Detection of CD107a Release Level During Tumor Cell KillingInduced by CAR-T19/20s Cells

1×10⁵ cells of effective cell CAR-T19/20s (CART-TN-OF-19 andCART-TN-LEU-19) were co-cultured respectively with 2×10⁵ of target tumorcells. The target cells are K562−CD19+, K562−CD20+, K562−CD19+CD20+, andK562 cells, Raji cells, Romas cells, respectively. At the same time, 3μl of Anti-Human CD107Ape was added to each well for staining and placedat 37° C., 5% CO 2 for 1 hour, and then 3 μl of 1% Golgistop was addedand placed at 37° C., 5% CO₂ for 3.5 hours. Next, 2 μl of CD8 FITC and1.5 μl of CD3APC were added to each well and incubated at 37° C., 5% CO₂for 30 min. 200 uL of FACS Buffer was added to each well and centrifugedat 300 g for 5 min. The liquid in the microplate was quickly discarded,and the remaining liquid was removed with absorbent paper. The plate waswashed with FACS Buffer again. The plate was stained with 7-AAD anddiluted with FACS Buffer (1:300). 200 uL of resuspended cells were addedto each well. The plate was detected by flow cytometry after 10 min,light avoided. The results were statistically analyzed.

The results are shown in FIG. 5. Both CART cells can well induce therelease of CD107a during tumor cell killing. Wherein, CART-TN-OF-19 havea slightly higher release of CD107a in the killing process and astronger killing effect than CART-TN-LEU-19.

Example 7 Inhibitory Effect of CAR-T19/20s on Transplanted Tumor Cellsin Mice

The tumor cells injected in animals are Raji. The tumor cells Raji carrya luciferase reporter gene (Raji expressing luciferase). In thisexperiment, tumor cells Raji were injected and grown in mice for oneweek, and then effector T cells were injected. The effector T cells weredivided into three groups: NT, CART-TN-LEU-19, and CART-TN-OF-19.

FIG. 6A shows the Protein L method for the detection of the transfectionefficiency of combined chimeric antigen receptor engineered T cellstargeting CD19CD20. The expression level of CD137 and the secretionlevel of IFNγ in the culture supernatant were the same as before. FIG.6A shows the TN-LEU-19, TN-OF-19 both have high transfection efficiency.FIGS. 6B and 6C show the expression levels of CD137 on the surface of Tcell membranes on the 10th day and the 18th day. Both TN-LEU-19 andTN-OF-19 can be expressed at high levels. FIG. 6D shows the secretionlevel of IFNγ in the culture supernatant on the 10th day. The resultsshow that the level in TN-OF-19 is significantly higher than that inTN-LEU-19.

On the 21st day, the expanded effector T cells (sent to the AnimalExperimental Center of Nanjing Medical University) were injected intothe mice through the tail vein, and then the fluorescence intensity ofthe mice (via IVIS fluorescence imaging) and the weight of the mice wererecorded every 7 days. The experiment was stopped on the 21st day, andthe statistical results were analyzed.

The results are shown in FIG. 6E. Both CART-TN-OF-19 and CART-TN-LEU-19CART cells can well inhibit the growth of mouse tumor cells comparedwith NT. FIG. 6E (left) shows the weight change of mice after theinjection of effector T cells into the three groups of mice. Comparedwith two types of CART cells CART-TN-OF-19 and CART-TN-LEU-19, the miceof NT showed a significant decrease in body weight and the mice in bothCART cell groups gained slightly more weight. FIG. 6E (right) shows theaverage fluorescence intensity of the three groups of mice. The resultsshow that the average fluorescence intensity of the mice in the NT groupincreased significantly, while the average of the fluorescenceintensities of the mice in the two CART cell groups decreasedsignificantly, and even difficult to detect.

FIG. 6F shows the IVIS imaging of fluorescence intensity in mice. Theresults showed that after the injection of effector T cells in the tailvein, the fluorescence intensities of the mice in the two CART cellgroups were very weak after the 7th day, while that of NT group was verystrong. The situation on 14th day was the same. The mice in NT group hadall died on the 21st day, while the mice in the two CART cell groupswere still growing normally.

Example 8 Screening and Functional Verification of CAR-T20.1, CAR-T20.2and CAR-T20.4

The construction and detection of CART cells were performed withreference to Examples 2, 3, 4, and 5.

First, full-length DNA was synthesized and cloned to achieve theconstruction of encoding plasmids. CAR-T 20.1, 20.2 and 20.4 weredesigned (the structures are shown in Table 1 and the sequences areshown in Table 2), and then the functional verification was performed.

PBMCs were thawed and infected to obtain CAR-T20s cells. Starting fromday 6, CAR-T20s cells can be taken for the corresponding activity assay.

0.5×10⁶ cells of CAR-T20s cell sample cultured 7 days were taken anddetected for the cell transfection efficiency by DNA copy number.

The results are shown in FIG. 7A. Compared to the positive control, theDNA copy number was not very high. Next, co-culture was performed, andCAR-T20s cells cultured for 7 days were used to detect the indicatorproteins CD137 and IFNγ of the cell activation level.

1×10⁵ of CAR-T20 cells cultured for 7 days were taken and culturedrespectively with CD20-positive cells Raji, Ramos and negative cellsK562, Karpas tumor cell line for 18 h with a ratio of 1:1. Then theexpression levels of CD137 on the surface of T cell membrane and thesecretion levels of IFNγ in the culture supernatant were detectedrespectively.

The results are shown in FIGS. 7B and 7C. CAR-T20.1, CAR-T20.2 andCAR-T20.4 constructed in this example are invalid.

Example 9 Screening and Functional Verification of CAR-T20.5, CAR-T20.6,CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10

The construction and detection of CART cells were performed withreference to Examples 2, 3, 4, and 5.

First, full-length DNA was synthesized and cloned to achieve theconstruction of encoding plasmids. CAR-T20.5, CAR-T20.6, CAR-T20.7,CAR-T20.8, CAR-T20.9 and CAR-T20.10 were designed (the structures areshown in Table 1 and the sequences are shown in Table 2), and then thefunctional verification was performed.

PBMCs were thawed and infected to obtain CAR-T20s cells. Starting fromday 6, CAR-T20s cells can be taken for the corresponding activity assay.

0.5×10⁶ cells of CAR-T20s cell sample cultured⁷ days were taken anddetected for the T cell transfection efficiency. Protein L method wasused to identify the expression level of CAR gene-encoded protein on thesurface of T cell membrane in CAR-T20s cells cultured for 7 days.

The results are shown in FIG. 8A. Compared with CAR-T20.1, amongCAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10,only CAR-T20.9 and CAR-T20.10 had a higher positive rate.

Next, co-culture was performed, and CAR-T20s cells cultured for 7 dayswere used to detect the indicator proteins CD137 and IFNγ of the cellactivation level. 1×10⁶ of CAR-T20s cells cultured for 7 days werecultured respectively with CD20-positive cells Raji, Ramos and negativecells K562, Karpas tumor cell line for 18 h with a ratio of 1:1. Thenthe expression levels of CD137 on the surface of T cell membrane and thesecretion levels of IFNγ in the culture supernatant were detectedrespectively.

The results are shown in FIGS. 8B and 8C. The results show that amongCAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10,only in CAR-T20.9 and CAR-T20.10, the expression levels of CD137 on thesurface of T cell membrane were activated and IFNγ was released in theculture supernatant. That is, CAR-T20.9 and CAR-T20.10 are effective.

Example 10 Screening and Functional Verification of CAR-T20.11,CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15 and CAR-T20.16

The construction and detection of CART cells were performed withreference to Examples 2, 3, 4, and 5.

First, full-length DNA was synthesized and cloned to achieve theconstruction of encoding plasmids. CAR-T20.11, CAR-T20.12, CAR-T20.13,CAR-T20.14, CAR-T20.15 and CAR-T20.16 were designed (the structures areshown in Table 1 and the sequences are shown in Table 2), and then thefunctional verification was performed.

PBMCs were thawed and infected to obtain CAR-T20s cells. Starting fromday 6, CAR-T20s cells can be taken for the corresponding activity assay.

0.5×10⁶ of CAR-T20s cells sample cultured for 7 days were taken anddetected for the T cell transfection efficiency. Protein L method wasused to identify the expression level of CAR gene-encoded protein on thesurface of T cell membrane in CAR-T20s cells cultured for 7 days.

The results are shown in FIG. 9A. Compared with CAR-T20.1, CAR-T20.9 andCAR-T20.10, the positive rates of CAR-T20.11, CAR-T20.12, CAR-T20.14 andCAR-T20.16 were high, while the positive rates of CAR-T20.13 andCAR-T20.15 were very low.

Next, co-culture was performed, and CAR-T20s cells cultured for 7 dayswere used to detect the indicator proteins CD137 and IFNγ of the cellactivation level. 1×10⁶ of CAR-T20s cells cultured for 7 days werecultured respectively with CD20-positive cells Raji, Ramos and negativecells K562, Molt-4 tumor cell line for 18 h with a ratio of 1:1. Thenthe expression level of CD137 on the surface of T cell membrane and thesecretion level of IFNγ in the culture supernatant were detectedrespectively.

The results are shown in FIGS. 9B and 9C. The results show that afterco-culture with CD20-positive Raji and Ramos, the expression levels ofCD137 on the surface of T cell membranes in CAR-T20.9, CAR-T20.10,CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14 and CAR-T20.16 wereactivated, and the releases were high, wherein CAR-T20.10 and CAR-T20.14were the highest, followed by the rest, only CAR-T20.15 showed noresponse. Correspondingly, the releases of IFNγ in the culturesupernatant were the same. The results showed that CAR-T20.10 andCAR-T20.14 had the best activity in this screening.

Example 11 Screening and Functional Verification of CAR-T20.17,CAR-T20.18 and CAR-T20.19

The construction and detection of CART cells were performed withreference to Examples 2, 3, 4, and 5.

First, full-length DNA was synthesized and cloned to achieve theconstruction of encoding plasmids. CAR-T20.17, CAR-T20.18 and CAR-T20.19were designed (the structures are shown in Table 1 and the sequences areshown in Table 2), and then the functional verification was performed.

PBMCs were thawed and infected to obtain CAR-T20s cells. Starting fromday 6, CAR-T20s cells can be taken for the corresponding activity assay.

0.5×10⁶ cells of CAR-T20s cell sample cultured for 7 days were taken anddetected for the T cell transfection efficiency. Protein L method wasused to identify the expression level of CAR gene-encoded protein on thesurface of T cell membrane in CAR-T20s cells cultured for 7 days.

The results are shown in FIG. 10A. Compared with CAR-T20.9, CAR-T20.12and CAR-T20.14, the positive rates of CAR-T20.17, CAR-T20.18 andCAR-T20.19 were higher, and the expressions thereof were well.

Next, co-culture was performed, and CAR-T20s cells cultured for 7 dayswere used to detect the indicator proteins CD137 and IFNγ of the cellactivation level. 1×10⁶ of CAR-T20s cells cultured for 7 days werecultured respectively with CD20-positive cells Raji, Ramos and negativecells Karpas-620, Molt-4 tumor cell line for 18 h with a ratio of 1:1.Then the expression levels of CD137 on the surface of T cell membraneand the secretion levels of IFNγ in the culture supernatant weredetected respectively.

The results are shown in FIGS. 10B and 10D. After co-culture withCD20-positive Raji, Ramos, the expression levels of CD137 on the surfaceof T cell membrane in CAR-T20.17, CAR-T20.18 and CAR-T20.19 wereactivated and released very high. Correspondingly, the releases of IFNγin the culture supernatant were the same, wherein the releases ofCAR-T20.17, CAR-T20.18 and CAR-T20.19 were very high. Subsequently, thedetection of LDH levels released by CAR-T20s cells for killing targetedtumors in vitro was performed. Specific experimental methods can bereferred to Example 5.

Target cells comprise CD20-positive Raji, Romas, and CD20-negativeMolt4. The final killing results showed that CAR-T20.17, CAR-T20.18, andCAR-T20.19 had strong killing effects on target cells CD20-positive Rajiand Romas.

The results of Examples 8-11 are summarized as follows:

The applicant of the present invention conducted a large number ofexperiments and screened out multiple CARs with good effects. Throughcomparison, it was found that 20.1, 20.2, 20.4, 20.5, 20.6, 20.7, 20.8and 20.15 were basically invalid, and 20.11, 20.12 and 20.13 had certaineffects, but the effects of them were less than that of 20.9, 20.10,20.14, 20.16, 20.17, 20.18 and 20.19, wherein the effects of 20.18 and20.19 were the best. Based on the above structures, CD20 Scfv and CD19scFv (FMC63) were tandemly used in a new bispecific chimeric antigenreceptor.

The structures of the chimeric antigen receptor in the CART cellsinvolved in Examples 8-11 are shown in Table 1 below, and the sequencesare shown in Table 2 below.

TABLE 1 Chimeric antigen receptors and structures thereof Change ofCAR-T Structure CAR- [CD8LS]- CD20(AY16076.1) [VL-Linker-VH]- T20.1 [hinge-CD8DE]-[4-1BB]-[CD3zeta] CAR- [CD8LS]- CD20(AY16076.1)[VL-Linker-VH]- T20.5  [hinge-CD8TM]-[4-1BB]-[CD3zeta] CAR- [CD8LS]-CD20(AY16076.1) [VH-Linker-VL]- T20.6  [hinge-CD8TM]-[4-1BB]-[CD3zeta]CAR- [CD8LS]- CD20(IDEC-C2B8) [VL-Linker-VH]- T20.7 [hinge-CD8TM]-[4-1BB]-[CD3zeta] CAR- [CD8LS]- CD20(IDEC-C2B8)[VL-Linker-VH]- T20.8  [hinge-CD8TM]-[4-1BB]-[CD3zeta] CAR- [CD8LS]-LEU16[VH-Linker-VL]-[hinge-CH2- T20.9  CH3-CD28TM] -[CD28]-[4-1BB]-[CD3zeta] CAR- [CD8LS]- LEU16[VL-Linker-VH]-[hinge-CH2- T20.10CH3-CD28TM] -[CD28] -[4-1BB]-[CD3zeta] CAR- [CD8LS]-LEU16[VL-Linker-VH]-[hinge-CH2- T20.11 CH3-CD8TM]-[4-1BB]-[CD3zeta] CAR-[CD8LS]- LEU16[VH-Linker-VL]-[hinge-CH2- T20.12 CH3-CD8TM]-[4-1BB]-[CD3zeta] CAR- [CD8LS]- Obinutuzumab[VH-Linker-VL]- T20.13[hinge-CH2-CH3-CD8TM] -[4-1BB]-[CD3zeta] CAR- [CD8LS]-Ofatuzumab[VH-Linker-VL]-[hinge- T20.14 CH2-CH3-CD8TM]-[4-1BB]-[CD3zeta] CAR- [CD8LS]- CD20(AY16076.1)[VH-Linker-VL]- T20.15[hinge-CH2-CH3-CD8TM] -[4-1BB]-[CD3zeta] CAR- [CD8LS]-Rituximab[VH-Linker-VL]-[hinge-CH2- T20.16 CH3-CD8TM] -[4-1BB]-[CD3zeta]CAR- [CD8LS]- LEU16[VH-Linker-VL]-[hinge-CH2-CH3- T20.17 CD28TM] -[CD28]-[4-1BB]-[CD3zeta](L235E N297Q) CAR- [CD8LS]-LEU16[VH-Linker-VL]-[hinge-CH2-CH3- T20.18CD8TM]-[4-1BB]-[CD3zeta](L235E N297Q) CAR- [CD8LS]-Ofatuzumab[VH-Linker-VL]-[hinge-CH2- T20.19 CH3-CD8TM]-[4-1BB]-[CD3zeta](L235E N297Q)

TABLE 2 Chimeric antigen receptors and sequences thereof SEQ ID CAR-TSequence NO. CAR-T20.1 MDIQLTQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGS 23SPKPWIYATSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSNPLTFGAGTKLEISSGGGGSGGGGSGDVMGVDSGGGLVQPGGSRKLSCAAPGFTFSSFGMHWVRQAPEKGLEWVAYISSPSSTLHYADRVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGPRDHVHRLLKTRLSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELEFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CAR-T20.4MALPVTALLLPLALLLHAARPDIQLTQSPAILSASPGEKVTMT 24CRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSNPLTFGAGTKLEISSGGGGSGGGGSGDVMGVDSGGGLVQPGGSRKLSCAAPGFTFSSFGMHWVRQAPEKGLEWVAYISSPSSTLHYADRVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGPRDHVHRLLKTRLSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELEFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR-T20.5MALPVTALLLPLALLLHAARPDIQLTQSPAILSASPGEKVTMT 25CRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSNPLTFGAGTKLEIGGGGSGGGGSGGGGSDVMGVDSGGGLVQPGGSRKLSCAAPGFTFSSFGMHWVRQAPEKGLEWVAYISSPSSTLHYADRVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGPRDHVHRLLTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELEFELGTFKTNDLQGSCRS CAR-T20.6MALPVTALLLPLALLLHAARPDVMGVDSGGGLVQPGGSRKL 26SCAAPGFTFSSFGMHWVRQAPEKGLEWVAYISSPSSTLHYADRVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGPRDHVHRLLGGGGSGGGGSGGGGSDIQLTQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSNPLTFGAGTKLEITRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELEFELGTFKTNDLQGSCRS CAR-T20.7MALPVTALLLPLALLLHAARPQIVLSQSPAILSASPGEKVTMT 27CRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSATRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELEFELGTFKTNDLQGSCRSCAR-T20.8 MALPVTALLLPLALLLHAARPQVQLQQPGAELVKPGASVKM 28SCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKTRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELEFELGTFKTNDLQGSCRSCAR-T20.9 MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKM 29SCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CAR-T20.10MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKM 30SCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CAR-T20.11MALPVTALLLPLALLLHAARPDIVLTQSPAILSASPGEKVTMT 31CRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CAR-T20.12MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKM 32SCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR CAR-T20.13MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKV 33SCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR CAR-T20.14MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLS 34CAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CAR-T20.15MALPVTALLLPLALLLHAARPGDVMGVDSGGGLVQPGGSR 35KLSCAAPGFTFSSFGMHWVRQAPEKGLEWVAYISSPSSTLHYADRVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGPRDHVHRLLKGGGGSGGGGSGGGGSDIQLTQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISTVEAEDAASYFCHQWSSNPLTFGAGTKLEIESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPRCAR-T20.16 MALPVTALLLPLALLLHAARPQVQLQQPGAELVKPGASVKM 36SCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR CAR-T20.17MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKM 37SCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR CAR-T20.18MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKM 38SCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY CAR-T20.19MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLS 39CAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR

Example 12 In Vivo Experiments in Mice of CART-TN-OF-19 andCART-TN-LEU-19

The inhibitory effects of CART-TN-OF-19 and CART-TN-LEU-19 cells ontransplanted tumor cells in mice was tested. The detection method wasperformed with reference to Example 7, and the control group was NT.

The results are shown in FIG. 11. Both CART cells can inhibit tumor cellexpansion well. The fluorescence intensities of mice in the CART cellgroups were very weak, while that of the NT group was very strong.Wherein, CART-TN-OF-19 can inhibit or kill tumor cells better thanCART-TN-LEU-19 in vivo.

Example 13 Phase I Clinical Trial of CART-TN-OF-19

After approval by the ethics committee, a total of 3 volunteers (numbersC001, C002, and C003) were conducted in Phase I clinical trials. The keycriteria for volunteer selection are as follows: the age should be 18-75years, having received more than 2 DLBCL treatment, PD after orineligible for auto-SCT, having not received anti-CD19 therapy, havingno active CNS, and having sufficient liver, kidney, heart, andhematopoietic functions.

The experimental process is shown in FIG. 12.

The clinical responses of each subject is shown in Table 3. The resultsshowed that the objective response rate (ORR) was 100%. After 4 weeks oftreatment, all three patients achieved partial remission (PR). 1 patientachieved complete remission (CR) at 8 w, and 2 patients achieved CR at 3m, and the clinical response is ongoing.

TABLE 3 Clinical responses of experimental subjects Parameter ShanghaiTongji Hospital Patient C001 C003 C004 Infusion time Nov. 20, 2019 Dec.20, 2019 Dec. 30, 2019 Response SPD Response SPD Response SPD Base-2203.12 1751.13 4956.14 line  4 w PR(CT) 862.86 PR(CT) 796.77 PR(CT)1645.45 (60.8%) (54.5%) (66.8%)  8 w CR(CT) 211.32 NE* PR(CT) 1501.39(90.4%) (69.7%) All LDi ≤1.5 cm 12 w NE* CR(PET/ CR(PET/ CT) CT) 15 wCR(PET/ CT) *NE because of COVID-19

The adverse events that occurred after treatment are shown in Table 4.The patients generally well tolerated safety profile. One of the threepatients had a grade 2 cytokine release syndrome, and two had a grade 1cytokine release syndrome. There were no death reported, no CRESreported. Cytopenia mostly related to Cy/Flu lymphodepletion.

It needs to be explained that the occurrence of a certain degree ofcytokine release syndrome after treatment also shows the effectivenessof CART treatment from the side, and no particularly serious cytokinerelease syndrome occurred in 3 patients. CART-TN-OF-19 has bettersecurity.

TABLE 4 Adverse reactions of subjects after treatment Parameter C001C003 C004 Cytokine release syndrome Grade 2 Grade 1 Grade 1Neurotoxicity No No No Neutropenia Grade 4 Grade 3 Grade 2Thrombocytopenia No No No Anemia Grade 3 No No

The results of the CAR copy number test are shown in FIG. 13, whichshows that the CAR copy number reached its peak (higher than 10⁵copies/microgram gDNA) in 14 days, and there was still a very high copynumber after 56 days.

The change in tumor size of an experimental subject is shown in FIG. 14,which shows that the tumor volume decreased significantly and almostdisappeared after 4 weeks of treatment.

All literatures mentioned in the present application are incorporatedherein by reference, as though each one is individually incorporated byreference. In addition, it should also be understood that, after readingthe above teachings of the present invention, those skilled in the artcan make various changes or modifications, equivalents of which falls inthe scope of claims as defined in the appended claims.

The invention claimed is:
 1. A bispecific chimeric antigen receptor(CAR), comprising an amino acid sequence set forth in SEQ ID NO: 16,wherein the bispecific CAR comprises an anti-CD20 antigen-binding regionand an anti-CD19 antigen-binding region.
 2. An immune cell comprising abispecific chimeric antigen receptor (CAR), wherein the bispecific CARcomprises an amino acid sequence set forth in SEQ ID NO: 16, and whereinthe bispecific CAR comprises an anti-CD20 antigen-binding region and ananti-CD19 antigen-binding region. .
 3. The immune cell of claim 2,wherein the immune cell is a T cell or a natural killer (NK) cell.
 4. Apharmaceutical composition comprising an immune cell which comprises abispecific chimeric antigen receptor (CAR), wherein the bispecific CARcomprises an amino acid sequence set forth in SEQ ID NO: 16, and whereinthe bispecific CAR comprises an anti-CD20 antigen-binding region and ananti-CD19 antigen-binding region.